Bacterial infections as a complication of surgery, prolonged hospitalization, accidents and other traumatic events, may lead to serious clinical symptoms such as sepsis, septic shock, inadequate organ perfusion, multiple organ failure and acute respiratory distress syndrome (ARDS). Despite advances in medicine over the past decade, an increase in the incidence of sepsis is evident with a mortality rate of 20 to 80%. The sepsis syndrome is initiated when micro-organisms bypass the natural defensive barriers of the body, such as skin and mucous membranes. If the immune system is unable to arrest the infection locally, the organism or its toxins may invade the circulation, where specific bacterial products elicit an inflammatory response that leads to the activation of an array of plasma proteins and cellular defense systems. Although mobilisation of the defence systems of the host is of paramount importance in combatting invading organisms, a cascade of events may simultaneously be triggered that can lead to irreversible tissue injury and organ dysfunction. Uncontrolled infections with Gram-negative bacteria such as Escherichia coli, Klebsiella spp., Neisseria spp., Pseudomonas aeruginosa, Salmonella spp, or Bordella spp. or the Gram-positive bacteria Staphylococcus aureus, Enterococcus spp., Streptococcus spp., Micrococcus luteus or Listeria monocytogenes give rise to a variety of clinical symptoms collectively referred to as the sepsis syndrome. The component of Gram-negative bacteria responsible for the initiation of the host response is termed endotoxin or lipopolysaccharide (LPS), a major glycolipid constituent of the outer membrane. In the circulation, LPS stimulates specific blood cells to produce endogenous mediators of inflammation termed cytokines such as tumor necrosis factor alpha (TNF-xcex1), interleukin-6 (IL-6) and interleukin-8 (IL-8) which have profound physiological effects on the organs and blood vessels of the body. Persistent stimulation of the cellular defence system by excessive LPS leads to overproduction of cytokines which activate a cascade of secondary inflammatory mediators eventually leading to blood vessel damage, circulatory and metabolic disturbances. The toxic component of the LPS molecule is the highly conserved Lipid A moiety which is sufficient to induce the pathophysiological changes characteristic of sepsis.
The prognosis of patients with endotoxemia would be considerably improved if the onset of sepsis could be detected at a sufficiently early stage in the disease process to enable effective treatment. Direct measurement of circulating endotoxins is of importance for the prediction of important clinical events such as bacteremia, septic shock and death. Clinically significant endotoxemia may go undetected by the currently available endotoxin assay, the Limulus amoebocyte lysate assay or LAL test, which has been shown to have serious limitations relating to sensitivity as well as to interference by plasma factors.
Current therapeutic options for Gram-negative bacterial sepsis are limited to anti microbial agents, hemodynamic support and management of sepsis-induced organ dysfunction. Although conventional antibiotic therapy is effective in halting the proliferation of susceptible micro-organisms, the massive release of LPS into the circulation by damaged bacteria may aggravate a septic episode. The relative importance of endotoxin release (endotoxemia) versus bacterial proliferation (bacteremia) during Gram-negative septic shock, however, has not fully been defined. Extensive clinical use of conventional antibiotics such as penicillins, cephalosporins and the like, in the treatment of bacterial infections during the past three decades, has resulted in a dramatic reduction in the efficacy of antibiotic therapy due to an alarming increase in the number of multi-drug-resistant bacteria.
Efforts to intervene directly in the pathophysiological mechanisms which underlie the septic process have yielded inconsistent and largely disappointing results. Anti-endotoxin monoclonal antibodies, anti-cytokine therapies and other anti-inflammatory strategies have proven not to be of sufficient benefit to warrant approval as standard adjunctive therapies for human sepsis. Because of the central role of LPS in the development of the sepsis syndrome, therapies designed to enhance the clearance or to neutralise the detrimental effects of endotoxin may prove beneficial. Therapy targeting the initial interactions of LPS is likely to be of most benefit when administered either early in the pathogenesis before widespread vascular injuries have taken place or prophylactically to high risk patients. However, since it is important to prevent further activation of inflammatory cells during the course of bacterial sepsis there may be a role for such treatment even after septic shock has become established. Characteristics of an anti-endotoxin reagent that would be desirable for therapeutic application include specific and avid binding to LPS concomitant with LPS-neutralizing activity, inherent systemic stability and low cytotoxicity.
A clinical syndrome indistinguishable from Gram-negative septic shock via the same endogenous mediators of inflammation in the absence of endotoxemia may be initiated by Gram-positive bacteria. In this instance, the initiation of the host response has been attributed to lipoteichoic acid (LTA), a major constituent of the outer membrane of Gram-positive bacteria. An additional cell-wall component common to both Gram-negative and Gram-positive bacteria that has been shown to induce cytokines in vitro, is the peptidoglycan and/or naturally occurring breakdown products of this macromolecular structure. Although LPS and LTA share few common structural features, one common physical property of these molecules is amphipathicity, a consequence of a specific orientation of negatively charged hydrophilic groups and hydrophobic side chains of long-chain fatty acid residues. On the basis of recent experimental evidence, a number of common steps in the pathway of cytokine induction by these toxic bacterial cell-wall components has been proposed.
Anti-microbial peptides are generally induced in animals in response to injury and infection. The synthesis of these factors of the innate or non-adaptive immune system may be induced by a variety of stimuli including Grain-positive and Gram-negative bacteria, fungi and viruses. Animal peptide antibiotics are generally small linear or cyclic basically charged molecules such as cecropins and defensins from mammalian and insect cells, magainin from frog skin, melittin from bee venom and tachyplesins from the horseshoe crab hemolymph which act on a rather broad spectrum of microbial organisms that often belong to the natural flora associated with the animal. The antibacterial activity of the linear class of cationic peptide antibiotics has been found to be dependent on the ability to form xcex1-helical structures which are capable of disrupting the integrity of the bacterial outer membrane (OM) by the formation of ion-channels in the lipid bilayer due to self-aggregation of peptide monomers in which the hydrophilic amino acid residues are oriented towards the interior of the membrane pore and the hydrophobic residues at the exterior interacting with phospholipid groups of the cell wall. The molecular basis of the integrity of the OM resides in the electrostatic linkage between the negatively charged Lipid A components of adjacent LPS molecules and divalent cations such as Mg2+ and Ca2+. Disruption of the cross-linkages, by displacement of these cations with positively charged entities of high affinity for LPS, was postulated to result in membrane destabilization. However, previous studies have also indicated that cationicity of an anti-microbial peptide alone is not the sole determinant for OM-permeabilizing activity, but that a specific configuration is essential for high affinity binding to LPS. In addition, since the Lipid A moiety of LPS is not easily accessible from the periphery of the cell by virtue of being submerged in the membrane, a further requirement of an effective LPS ligand could be small size for effective membrane penetration.
A number of naturally occurring proteins and polypeptides have been reported to bind and neutralize LPS. These include polymyxin B (PmB), Limulus-anti-LPS factor (LALF), the human neutrophil-derived CAP18 and bactericidal/permeability-increasing protein (BPI). PmB and LALF detoxify LPS in vitro and afford protection against endotoxin-mediated lethality in experimental animals, but toxicity precludes their clinical use against Gram-negative bacterial infections. The CAP18 protein inhibits LPS responses in vitro but currently available data are not sufficient for assessment as a potential therapeutic agent in the treatment of sepsis. Limitations to the therapeutic utility of the BPI protein or the truncated recombinant derivatives rBPI23 or rBPI21 in clinical endotoxic shock, are the rapid clearance from the circulation and lowered efficacy against complex endotoxins. Synthetic peptide derivatives incorporating the potential LPS-binding domains of LALF, CAP18 or BPI have all shown unfavourable properties such as diminished anti-microbial activity and lowered affinity for Lipid A with respect to the parent molecules.
The applicants have derived a new generation of LPS-binding peptides termed bactericidal peptides (BP) by means of molecular modelling and rational design techniques. General properties included in the design of the peptides were restricted size, unique conformational and chemical characteristics, solubility and low cytoxicity. Specific characteristics included in the structure of the peptides were the presence of multiple sequence elements constituting potential LPS-binding domains presented in a specific conformation for optimal binding (high avidity) to the lipid A component of LPS. The peptides can be synthesized by solid-state chemistry with Fmoc (9-fluorenylmethoxy-carbonyl) amino acid derivatives, purified to homogeneity by reverse-phase high-pressure liquid chromatography and verified by analytical HPLC, amino acid analysis and mass-spectrometry in a manner known per se. Any known methodology of peptide synthesis can be applied and is readily available to a person skilled in the art.
Biochemical and biological characterisation of the synthetic peptides or biotinylated derivatives demonstrated:
1) Potent anti-microbial activities against Gram-negative as well as Gram-positive bacteria
2) The ability to bind specifically to the Lipid A components of heterologous endotoxins with high affinity relative to Polymyxin B, an established high affinity endotoxin binder.
3) The ability to complex with and precipitate LPS from solution.
4) The ability to detect endotoxin concentrations of  less than 0.1 pg/ml in diluted plasma.
5) The ability to neutralize endotoxin-mediated cytokine production in whole blood ex vivo.
6) The ability to prevent septic shock in mice challenged with lethal doses of endotoxin.
7) The ability to prevent septic shock in mice challenged with lethal doses of live pathogenic bacteria.
As a consequence of the unique properties of the synthetic peptides applications could include:
1) Treatment of topical and systemic infections by multi-drug-resistant bacteria with peptide alone or in combination with conventional antibiotics.
2) Quantitative removal of endotoxins from physiological and pharmaceutical solutions.
3) Development of a new endotoxin assay, termed the Endotoxin Inhibition ELISA (EIE) on a commercial basis for clinical diagnostic use with the use of biotinylated peptide derivatives.
4) Prevention of sepsis by prophylactic use of peptides of the invention after surgery of high risk patients.
5) Treatment of the septic shock syndrome in mammals including humans.
Considering the general membrane destabilizing properties of amphipathic xcex1-helical peptides as well as the demonstrated antitumor as well as the predicted antifungal, antiviral and anti-inflammatory properties, other possible applications of the synthetic peptides of the invention include:
6) Treatment of the infectious disease caused by the systemic merezoite forms of the malaria parasite Plasmodium spp.
7) Treatment of other parasitic diseases such as Trypanosomiosis.
8) Treatment of topical and systemic malignancy.
9) Treatment of fungal infections.
10) Treatment of viral infections.
11) Treatment of inflammation.
Accordingly the primary object of the invention is to employ the novel peptides in the further development of a new endotoxin assay on a commercial basis for clinical diagnostic use.
It is also an object of the invention to provide novel prophylactic peptides for use in the prevention of septic shock.
It is also an object of this invention to provide novel therapeutic peptides for use in the treatment of septic shock.
It is also an object of the invention to provide novel peptides for the quantitative removal of endotoxins from pharmaceutical solutions.
It is also an object of the invention to provide novel therapeutic peptides for the treatment of infectious diseases in general.